A primary cell culture has been developed from rat mammary glands which is suitable for the study of hormonal regulation of lipoprotein lipase activity. Enzyme digests of mammary glands from rats in midpregnancy are separated into five density fractions on Ficoll gradients. The cells of each fraction are maintained in a complex hormone-supplemented medium. Lipoprotein lipase activity of each of the cell fractions is initially low but that of Fractions I and II, which accumulate large lipid droplets, increases 10-20 fold between the ninth and twelfth day of culture. Nutritionally selected cells of Fractions I and II produce lipoprotein lipase in the presence of prolactin plus a limited combination of hormones. These preliminary findings suggest that in culture a specific epithelial cell type in mammary gland produces lipoprotein lipase in response to hormones.